Angiogenesis is the development of new vasculature from preexisting blood vessels and/or circulating endothelial stem cells (Asahara et al., Science, 275,(5302):964–967, 1997; Springer et al., Mol. Cell, 2(5):549–558, 1998; Folkman and Shing, J. Biol. Chem., 267:10931–10934, 1992). Angiogenesis plays a vital role in many physiological processes, such as embryogenesis, wound healing and menstruation. Angiogenesis is also important in certain pathological events. In addition to a role in solid tumor growth and metastasis, other notable conditions with an angiogenic component are arthritis, psoriasis and diabetic retinopathy (Hanahan and Folkman, Cell, 86(3):353–364, 1996; Fidler and Ellis Cell 79(2):185–188, 1994).
Angiogenesis is regulated in normal and malignant tissues by the balance of angiogenic stimuli and angiogenic inhibitors that are produced in the target tissue and at distant sites (Fidler et al., Cancer J. Sci. Am., 4 Suppl 1: S58–66, 1998; McNamara et al. Br. J. Surg., 85(8):1044–1055, 1998). Vascular endothelial growth factor (VEGF) is an important factor driving angiogenesis or vasculogenesis in numerous physiological and pathological processes, including wound healing (Frank et al., J. Biol. Chem., 270:12607–12613, 1995; Burke et al., Biochem. Biophys. Res. Comm., 207:348–354, 1995), diabetic retinopathy (Alon et al., Nature Med., 1:1024–1028, 1995; Malecaze et al., Arch. Ophthalmol, 112:1476–1482, 1994), psoriasis (Detmar et al., J. Exp. Med., 180:1141–1146, 1994), atherosclerosis (Inoue et al., Circulation, 98(20):2108–16, 1998), rheumatoid arthritis (Harada et al., Scandinavian J. Rheumatol., 27(5):377–80, 1998; Nagashima et al., Clin. Exp. Immunol., 116(2):360–5, 1999), solid tumor growth (Plate et al., Int. J. Cancer, 59:520–529, 1994; Claffey et al., Cancer Res., 56:172–181, 1996).
VEGF is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations. VEGF is a primary stimulant of the development and maintenance of a vascular network in embryogenesis. It functions as a potent permeability-inducing agent, an endothelial cell chemotactic agent, an endothelial survival factor, and endothelial cell proliferation factor (Thomas, J. Biol. Chem., 271:603–606, 1996; Neufeld et al., FASEB J., 13(1):9–22, 1999). Its activity is required for normal embryonic development as targeted disruption of one or both alleles of VEGF results in embryonic lethality (Carmeliet et al., Nature, 380(6573):435439, 1996; Ferrara et al., Nature, 380(6573):439–442, 1996).
A wide variety of cells and tissues produce VEGF, which exists in at least five isoforms (121, 145, 165, 189, and 206 amino acids) that are splice variants encoded by the same gene (Houck et al., Molec. Endo., 5(12):1806–1814, 1991; Ferrara et al., J. Cell. Biochem., 47:211–218, 1991; Tischer et al., J. Biol. Chem., 266:11947–11954, 1991). The two smaller isoforms, 121 and 165, are secreted from cells (Houck et al., Molec. Endo., 5(12):1806–1814, 1991; Anthony et al., Placenta, 15:557–61, 1994). Secreted VEGF is an obligate dimer of between 38–46 kDa in which the monomers are linked by two disulfide bonds.
VEGF dimers bind with high affinity to two well-characterized receptors, VEGFR1 (FLT-1) and VEGFR2 (KDR/Flk-1), which are selectively expressed on endothelial cells (Flt-1 and Flk-1 are the mouse homologues). The Kd of VEGF binding to VEGFR1 and VEGFR2 is 15–100 pM and 400–800 pM, respectively (Terman et al, Growth Factors, 11(3):187–195, 1994). A recently identified third cell surface protein, neuropilin-1, also binds VEGF with high affinity (Olander et al., Biochem. Biophys. Res. Comm., 175:68–76, 1991; De Vries et al, Science, 255(5047):989–991, 1992; Terman et al., Biochem. Biophys. Res. Comm., 187:1579–1586, 1992; Soker et al., Cell, 92(6):735–745, 1998).
VEGFR1 and VEGFR2 are members of the Type III receptor tyrosine kinase (RTK III) family that is characterized by seven extracellular IgG-like repeats, a single spanning transmembrane domain, and an intracellular split tyrosine kinase domain (Mustonen and Alitalo, J. Cell Biol., 129:895–898, 1995). Until very recently, VEGFR1 and VEGFR2 were thought to be almost exclusively expressed on endothelial cells (Mustonen and Alitalo, J. Cell Biol., 129:895–898, 1995). Although VEGFR1 and VEGFR2 have been reported to have different functions with respect to stimulating endothelial cell proliferation, migration, and differentiation (Waltenberger et al., J. Biol. Chem., 269(43):26988–26995, 1994; Guo et al., Biol. Chem., 270:6729–6733, 1995), the precise role that each receptor plays in VEGF biology and endothelial cell homeostasis has not been clearly defined.
Recent studies using knockout mice have shown each of VEGF, VEGFR1 and VEGFR2 to be essential for vasculogenesis, angiogenesis and embryo development (Fong et al., Nature, 376:66–70, 1995; Shalaby et al., Nature, 376:62–66, 1995; Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95(16):9349–9354, 1998). In studies of lethal knockouts, the phenotypes associated with the lack of each receptor were different. Targeted disruption of VEGFR2 resulted in an embryo that lacked endothelial cell differentiation and failed to form yolk sac blood islands or go through vasculogenesis (Shalaby et al., Nature, 376:62–66, 1995). VEGFR1 null mutants showed impaired vasculogenesis, disorganized assembly of endothelial cells, and dilated blood vessels (Fong et al., Nature, 376:66–70, 1995; Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95(16):9349–9354, 1998). VEGFR1 evidently has a vital biological role.
VEGFR1 has a higher affinity for VEGF than VEGFR2, although it has a lower tyrosine kinase activity. This suggests that the extracellular domain of VEGFR1 is particularly important. This hypothesis was strongly supported by results from studies in knockout mice in which the tyrosine kinase domain of VEGFR1 was deleted while leaving the VEGF binding domain intact (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95(16):9349–9354, 1998). The VEGFR1-tyrosine kinase deficient embryos developed normal blood vessels and survived to term (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95(16):9349–9354, 1998).
In addition to the earlier knockouts (Fong et al., Nature, 376:66–70, 1995; Shalaby et al., Nature, 376:62–66, 1995), the Hiratsuka et al. (1998) studies indicate that VEGFR 1 has a vital biological role. However, tyrosine kinase signaling does not seem to be the critical factor. It is interesting to note that macrophages from the VEGFR1 knockout mice did not exhibit VEGF-induced chemotaxis (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95(16):9349–9354, 1998; incorporated herein by reference), thereby implicating VEGFR I as the receptor responsible for mediating this important biological response to VEGF.
Certain groups have reported VEGFR2 to be the dominant signaling receptor in VEGF-induced mitogenesis, and permeability (Waltenberger et al., J. Mol Cell Cardiol., 28(7):1523–1529, 1994; Zachary, Exp. Nephrol., 6(6):480–487, 1998; Korpelainen and Alitalo, Curr. Opin. Cell Biol., 10(2):159–164, 1998). The role of VEGFR1 in endothelial cell function is much less clear, although functions in macrophage migration and chemotaxis were documented in the Hiratsuka et al. (1998) studies discussed above.
Clauss et al. (1996; incorporated herein by reference) also reported that VEGFR1 has important roles in monocyte activation and chemotaxis. In fact, cells of the macrophage/monocyte lineage express only VEGFR1, which is the receptor responsible for mediating monocyte recruitment and procoagulant activity (Clauss et al., J. Biol. Chem., 271(30):17629–17634, 1996). VEGF binding to VEGFR1 on monocytes and macrophages also acts by raising intracellular calcium and inducing tyrosine phosphorylation (Clauss et al., J. Biol. Chem., 271(30):17629–17634, 1996).
Binding of the VEGF dimer to the VEGF receptor is believed to induce receptor dimerization. Dimerization of the receptor then causes autotransphosphorylation of specific tyrosine residues, Y801 and Y1175, and Y1213 and Y1333 on the intracellular side of VEGFR2 and VEGFR 1, respectively. This leads to a signal transduction cascade, which includes activation of phospholipase Cγ(PLCγ) and phosphatidylinositol 3-kinase (PI3K) and an increase in intracellular calcium ions (Hood and Meininger, Am. J. Physiol., 274(3 Pt 2):H1054–1058. 1998; Hood et al., J. Biol. Chem., 273(36):23504–23508, 1998; Kroll and Waltenberger, Biochem. Biophys. Res. Commun., 252(3):743–746, 1998).
The intracellular events further downstream in VEGF-induced signaling are less clear, although a number of groups have shown that nitric oxide (NO) is produced after VEGF activation of VEGFR2 (Hood and Meininger, Am. J. Physiol., 274(3 Pt 2):H1054–1058. 19981998; Hood et al., J. Biol. Chem., 273(36):23504–23508, 1998; Kroll and Waltenberger, Biochem. Biophys. Res. Commun., 252(3):743–746, 1998). Activation of VEGFR2, but not VEGFR1, by VEGF has also been shown to activate Src and the Ras-MAP kinase cascade, including the MAP kinases, ERK 1 and 2 (Kroll and Waltenberger, Biol. Chem., 272:32521–7, 1997).
The role of VEGFR1 in endothelial cell function is much less clear, particularly as Flt-1 tyrosine kinase-deficient mice are viable and develop normal vessels. It has been suggested that the main biological role of VEGFR1 on endothelial is as a non-signaling ligand-binding molecule, or “decoy” receptor that might be required to present VEGF to VEGFR2.
The connection between VEGF and pathological angiogenic conditions has prompted various attempts to block VEGF activity. These include the development of certain neutralizing polypeptides against VEGF. Polypeptides against VEGF receptors have also been described, such as described in U.S. Pat. Nos. 5,840,301 and 5,874,542 and, subsequent to the present invention, in WO 99/40118. U.S. Pat. Nos. 5,840,301 and 5,874,542 indeed suggest that blocking VEGF receptors rather than VEGF itself is advantageous for various reasons.
Soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors have also been reported (Saleh et al., Cancer Res., 56:393–4011996; Cheng et al., Proc. Natl. Acad. Sci. USA, 93:8502–8507, 1996; each incorporated herein by reference).